Collecting mitochondrial genome sequences from museum collections has garnered increasing attention in recent years. Currently, several sequencing strategies have been proposed for this purpose, including direct DNA pooling, DNA labeling, and mtDNA enrichment. It is important to understand the advantages and limitations of these methods before embarking on large-scale sequencing efforts. However, there is currently no comprehensive comparison and evaluation of the performance of these sequencing methods. In this study, we evaluated the performance of four different strategies for sequencing mitogenomes by comparing reagent expense, experiment time, bioinformatics time, and the length and integrity of the obtained mitochondrial genomes. The four sequencing strategies mainly differ in: (1) whether to label the DNA of each specimen and (2) whether to perform mtDNA enrichment. To reduce experiment difficulty and expenses, we presented an "in-situ" DNA labeling protocol for cost-effectively ligating barcode sequences to DNA samples and used PCR-generated baits for mtDNA enrichment. We found that the widely adopted "direct pooling" sequencing strategy is the fastest but performs the worst among the four methods we compared. Of the 48 lepidopteran specimens analyzed, the average length of mitochondrial sequences obtained per sample is less than 1000 bp. The most complicated sequencing strategy, which labels the DNA of each specimen and performs mtDNA enrichment, performs the best, producing mitogenome sequences of > 11 kb per sample, with 14 mitogenomes being (almost) complete. We hope that our comparisons will assist researchers in selecting suitable strategies for large-scale sequencing of mitogenomes from museum specimens, thereby advancing museomics.
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